Genome Wide Regulation Flashcards
GeneArrays
used to measure the amount of mRNA from all the genes in an organism
RNA-seq
Uses next generation DNA sequencing to determine transcriptome
2-D gels
used to measure the amount of proteins expressed
Mass-spectroscopy
identifies proteins
Gene Arrays
- Each gene of a sequenced organism is fixed to a slide in an ordered “array”
- the army is then probed with mRNA from your cell (two conditions)
Gene Array Technique
- RNA Isolation
- cDNA Generation
- Labeling of Probe
- Hybridization to Array
- Imaging
Campylobacter has how many genes
1600
Making the array
- genes or portions of genes are spotted in duplicate on glass slides by robots.
Probing the array
- microarrays are good at comparing transcription profiles between two different conditions
- First, you make cDNA copies of the RNA with reverse transcriptase
- cDNA from one condition is labeled red dye
- The other condition is labeled with green dye
- Both sets are then hybridized to the array, and a machine detects the color.
Gene Arrays
- then probed the away
- each gene on there twice
- yellow-both genes expressed
Heat Map
- shows how much red versus gene per green
- repeat experiment and swap dyes looking for exact opposite effect
- fold changes never really greater than 3-4
- only so green.
RNA seq
- total RNA
- eukaryote (enrich mRNA by oligo(dT))
- prokaryote (remove RNA by kit)
- RNA fragmentation
- Random hexameter primed cDNA synthesis
- Ends repairment, adapter ligation, and PCR amplification
- Hiseq 2000 91PE or 101PE sequencing
Flagella discussion
- RpoN mutant
- RNA isolated from both conditions
- DNA digested by DNase
- rRNA depleted
- cDNA made with random primers
- RpoN required for Flagella (class II genes)
- cjc242 is a host invasion space gene that uses hook apparatus to invade host cells
Proteomics
the study of all the proteins in a cell
advantages of proteomics
detects non-transcriptional control, can be cheaper
disadvantage of proteomics
cheap methods miss low abundance proteins
1st dimension of 2D electrophoresis
- separates by charge (isoelectric focusing)
- the charge on a protein depends on PH
- isolelectric point is pH where they have no charge
- proteins with positive charge will migrate toward negative electrode until they lose enough protons to lose charge
2nd dimension of 2D electrophoresis
- separates by size (SDS PAGE)
- each spot corresponds to size
Ahpc
highly expressed protein
NapA
compensates for loss of Ahpc
determining the spots
- spots that are regulated are cut out of the gel and extracted
- to determine protein identity:
- Do N-terminal sequencing
- determine the mass of protein by mass spectrometry.
Tryptic Digest
- trypsin cuts proteins after argentine and lysine residues
- excise the spot, treat the spot with trypsin to cut it up
- weight fragments by mass spectroscopy
LC-Maldi TOF
- Tags differ by 1 amu
- mass to charge ration (M/Z) is determined by the amount of time it takes to get to the detector - Time of Flight
- keep the charge 1, you get the mass
- time to get from pusher to detector tells you how big
- fly for about 12 feet
Mass spectrum
every fragment that comes through we weigh
